Name: A2780 Cells Genomic DNA Isolation and Analysis
Uploaded: 2023-04-21T13:20:00
Description: Watch this Scientific Journal Video about A2780 Cells Genomic DNA Isolation and Analysis at JoVE.com

The video outlines a detailed protocol for extracting genomic DNA from A2780 cells. It covers the use of lysis buffer, RNase, and proteinase K, followed by phenol-chloroform extraction and precipitation with sodium chloride and ethanol. The process ensures high-quality DNA suitable for analysis of telomere restriction fragments.

a2780 cells genomic dna isolation and analysis

Quantification de la longueur des télomères à l’aide de la détection par chimiluminescence non radioactive

Telomeres are the repetitive DNA sequences present at the end of chromosomes. They have tandem repeats of TTAGGG and maintain genome integrity by protecting the chromosome from both fraying and the end replication problem, which means that part of the 3&#39; overhang is unable to be replicated by DNA polymerase1,2. Short telomeres lead to chromosomal abnormalities in cells, due to which cells become permanently arrested in a stage called replicative senescence3. Short telomeres also cause a host of other problems, such as mitochondria dysfunction4,5 and cell dysfunction.
DNA telomeric repeats are lost as and when the cell divides, with an average loss of 25 to 200 bp per year6, resulting in cellular senescence after a certain number of divisions6. Aging is associated with a higher frequency of comorbidities, which is marked by a shortening in telomere length7. Telomere restriction fragment (TRF) analysis, as described by Mender, is a very expensive method8. Because of this, it is not implemented while quantifying telomere length in most studies.
Presently, the majority of epidemiological studies employ quantitative polymerase chain reaction (qPCR)-based measurements of telomere length. However, the qPCR-based method is a relative measurement method, as it measures the ratio between telomeres and single-copy gene amplification products, and not absolute telomere length. Telomere length measurement using the TRF protocol is the gold standard method, as it can measure telomere length distribution in the sample and measurements can be expressed in absolute values in kilobases (kb). However, its use is limited because it is cumbersome, labor-intensive, and costly. Here, we present an optimized protocol for telomere length measurement using chemiluminescence-based TRFs.
TRF analysis includes seven major steps: 1) culturing of cells for genomic DNA extraction, 2) genomic DNA extraction using the phenol:chloroform:isoamylalcohol (P:C:I) method, 3) restriction digestion of genomic DNA, 4) agarose gel electrophoresis, 5) Southern blotting of the restriction digestion DNA fragment, 6) hybridization and detection via chemiluminescence-the immobilized telomere probe is visualized by a highly sensitive chemiluminescent substrate for alkaline phosphatase, disodium 2-chloro-5-(4-methoxyspiro[1,2-dioxetane-3,2&#8242;-(5-chlorotricyclo[3.3.1.13.7]decan])-4-yl]-1-phenyl phosphate (CDP-Star)-and 7) analysis for obtaining mean telomere length and range information from these telomeric smears.

Pleins feux sur l’auteur : Optimisation des paramètres de performance du dosage de la longueur des télomères TAGGG  

Optimisation des paramètres de performance du test de longueur des télomères TAGGG

The scope of our research involves measuring telomere length differences across various study groups. The questions we are trying to answer revolve around diseases of physiological conditions and how they may affect telomere length and vice versa. The technology is currently used to advance research in the field of telomere length biology are nanopore sequencing and more recently CRISPR Cas based measurement.Current experimental challenges are accurately measuring absolute telomere length and the changes in it. The same sample could give varying results when different methods of measuring telomere length are used. The research gap we address is the lack of a standard method to measure telomere length.Telomere restriction fragment analysis or TRF, is still the standard against which all new techniques are evaluated. Also, our optimized protocols helps in processing a higher number of samples with limited resources. Our findings will help advance research in the field of telomere length biologic by providing a reliable and relatively economical way to compare newer methods to the gold standard, which is TRF.Our laboratory will focus on the effects of small molecule inhibitors on telomere length. We will also study the impact that lifestyle diseases and telomere length have on on each other.

Watch this Scientific Journal Video about A2780 Cells Genomic DNA Isolation and Analysis at JoVE.com

A2780 Cells Genomic DNA Isolation and Analysis  

A2780 Isolement et analyse de l’ADN génomique des cellules

Commencez par ajouter 500 microlitres du tampon de lyse à la pastille de cellule A2780 précédemment obtenue, et mélangez doucement à l’aide d’une pointe coupée d’un diamètre d’ouverture minimal de deux millimètres. Ajouter 20 microgrammes par millilitre de RNase fraîchement préparée et mélanger par inversion douce. Incuber à 37 degrés Celsius pendant 30 minutes.À la fin de l’incubation, ajouter la protéinase K jusqu’à une concentration finale de 100 microgrammes par millilitre et retourner doucement 10 fois. Incuber à nouveau à 55 degrés Celsius pendant une heure en inversant le tube toutes les 10 minutes. Pipeter 500 microlitres de réactif phénol:chloroforme:alcool isoamylique dans le tube et retourner doucement le tube environ 20 fois.Centrifuger à 9, 391 G pendant 15 minutes à température ambiante. Pipeter la couche visqueuse aqueuse supérieure dans un nouveau tube. Ajouter un volume égal de chloroforme et mélanger par inversion douce.Après avoir centrifugé le tube une fois, recueillir la couche aqueuse. Ajouter une quantité appropriée de cinq molaires de chlorure de sodium dans le tube pour augmenter la concentration de 0,2 mole. Ajouter deux volumes d’éthanol à 100% dans le tube et l’inverser doucement 20 fois.Centrifuger à 15 871 g pendant cinq minutes à température ambiante. Après avoir jeté le surnageant, ajoutez 500 microlitres d’éthanol à 70%. Centrifuger à nouveau dans le même état, puis jeter le surnageant.Une fois la pastille séchée à l’air, ajoutez 50 microlitres d’eau stérile sans nucléase et laissez-la se réhydrater. Ensuite, mélangez l’ADN en pipetant à l’aide de l’embout coupé. Utilisez la spectrophotométrie UV pour mesurer la concentration d’ADN.Après avoir digéré l’ADN, séparez-le à l’aide d’agarose à 0,8%. Plonger le gel d’agarose dans une solution d’acide chlorhydrique 0,25 molaire pendant 10 minutes à température ambiante en agitant doucement. Ensuite, rincez le gel deux fois avec de l’eau distillée.Pour dénaturer l’ADN, immergez le gel dans une solution d’hydroxyde de sodium et de chlorure de sodium. Ensuite, rincez le gel deux fois avec de l’eau distillée. Pour neutraliser l’ADN comme démontré, submerger le gel dans une solution d’acide chlorhydrique 0,5 molaire et trois molaires de chlorure de sodium à pH sept.L’ADN génomique de l’extracteur a montré une bonne intégrité, indiquant son utilisation pour le traitement ultérieur en aval des fragments de restriction des télomères.

Research

JoVE Journal

Cancer Research

A2780 Cells Genomic DNA Isolation and Analysis

Optimization of Performance Parameters of the TAGGG Telomere Length Assay

Telomeres are repetitive sequences which are present at chromosomal ends; their shortening is a characteristic feature of human somatic cells. Shortening ...